KR101733302B1 - Molecular marker for selecting watermelon line or cultivar resistant to powdery mildew and use thereof - Google Patents

Abstract

More particularly, the present invention relates to a SNP marker composition, a primer set, and a method for distinguishing resistance to watermelon mildew disease by using the SNP marker composition and primer set for distinguishing resistance to watermelon mildew disease will be.
In the development of resistant strain and strain of watermelon pollen resistant strain derived from PI 254744, by providing the resistance marker of watermelon powdery mildew according to the present invention, DNA of young plant or seed can be extracted without direct disease in order to confirm resistance.

Description

[0002] Molecular markers for the identification of resistance to watermelon powdery mildew and their use [0002] Molecular markers for selecting watermelon line or cultivar resistant to powdery mildew and use thereof [

More particularly, the present invention relates to a SNP marker composition, a primer set, and a method for distinguishing resistance to watermelon mildew disease using the SNP marker composition and primer set for distinguishing resistance to watermelon mildew disease, a strain or a variety, ≪ / RTI >

Currently, sequencing analysis of plants is rapidly proceeding due to the development of the sequencing method and cost reduction. In the case of poultry and plants, the full-length sequence of cucumber (2009), melon (2012) and watermelon (2013) has recently been published. Recently, a high-density genetic map based on the genome sequence has been created through genome sequence analysis. Due to the acquisition of single nucleotide polymorphism (SNP) information, not only the classification of various plant lines and cultivars, but also the utilization as molecular markers are increasing. In particular, the crops are infected with various diseases and their yields and quality are continuously deteriorated, and damage due to overuse of pesticides for controlling them is continuously increasing. The most desirable method for solving this problem is a disease resistance system induction method in which a resistant genome region of a wild type including a disease resistance gene that does not exist in an existing cultivar is introduced into a cultivar through crossing. In general, the disease-resistant genome region is introduced through backcross, and the disease-resistant system can be generalized through continuous inoculation. In recent years, according to the sequencing of the entire plant sequence, the existing gene map has been densified, and thus the trait-related markers obtained by quantitative trait loci (QTL) analysis related to a specific trait are also tightly linked marker). Candidate genes for specific genomic regions that confer disease resistance have also been found.

On the other hand, powdery mildew is an absolute active parasite, which is frequent in watermelon and other plants and plants. The symptoms are slightly different depending on the crops, but most of them are stalks, leaves, and foliage-like fungi. It inhibits respiration and reduces anabolic and hyperpigmentation, resulting in rapid aging with necrosis of diseased tissues, thus greatly affecting the cultivation and production of crops such as watermelon (Citrullus lanatus, watermelon).

To date, such powdery mildew control has relied mainly on chemical pesticides such as diphenoconazole, benomyl, and mangochi. However, the use of such chemical pesticides eventually increases the resistance of the powdery mildew to increase the resistance of the pesticide, Consumers are avoiding the use of pesticides and vegetables because of the high residual pesticide detection.

Therefore, breeding of watermelon resistant to powdery mildew is an urgent situation, and studies have been made, but it is still not enough.

DISCLOSURE OF THE INVENTION The object of the present invention is to solve the above-mentioned problems in the prior art. The object of the present invention is to provide a watermelon powder resistance QTL analysis in a segregation group in which citrus fruits (Citrullus lanatus, watermelon) commercial line SBB and wild type PI 254744 are cross- And to provide a molecular marker capable of discriminating resistance against powdery mildew caused by the watermelon PI 254744 genome based on the watermelon genome region obtained by the method.

Another object of the present invention is to provide a method for distinguishing resistance to watermelon mildew disease by using the molecular marker.

It is another object of the present invention to provide a method for breeding watermelon having resistance to powdery mildew.

However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

In order to achieve the above object,

A polynucleotide sequence consisting of 8-100 contiguous bases comprising a single nucleotide polymorphism (SNP) located in position 553 within the nucleotide sequence of SEQ ID NO: 1, or a complementary polynucleotide sequence thereof;

A polynucleotide sequence consisting of 8-100 contiguous bases comprising a single nucleotide polymorphism (SNP) located in position 553 within the nucleotide sequence of SEQ ID NO: 2, or a complementary polynucleotide sequence thereof; And

A polynucleotide sequence consisting of 8-100 contiguous bases comprising a single nucleotide polymorphism (SNP) located in the 549th nucleotide in the nucleotide sequence of SEQ ID NO: 3 or a complementary polynucleotide sequence thereof And a complementary polynucleotide sequence thereof selected from the group consisting of SEQ ID NO:

The present invention also provides a set of primers for identifying a pollenucleotide sequence or a pollen-resistant pollenucleotide sequence capable of amplifying a complementary polynucleotide sequence thereof.

In one embodiment of the present invention, the primer set comprises a primer set consisting of SEQ ID NOS: 4 and 5; A primer set consisting of SEQ ID NOS: 6 and 7; And a set of primers consisting of SEQ ID NOS: 8 and 9.

In addition, the present invention provides a PCR kit for HRM analysis for discriminating resistance to watermelon mildew disease including the primer set.

Further, according to the present invention,

(1) performing PCR by mixing the PCR kit with whole DNA samples extracted from watermelons; And

(2) performing a high resolution melting (HRM) analysis using the amplified sample of step (1).

In addition, the present invention provides a method of breeding watermelon having a fungus resistance comprising the following steps:

(a) obtaining interspecific F1 by interspecific hybridization of resistant rice wild type PI 254744 and cultivated SBB or 45NC;

(b) obtaining a population of BC4F1, which is a species of watermelon powdery mildew resistant species, through crossing the interspecies F1 with SBB or 45NC again (F1 × SBB or F1 × 45NC); And

(c) self-moisturizing the BC4F1.

The present invention has the advantage of extracting the DNA of young plants or seeds without the need of direct inoculation in the development of resistant strains and varieties of the watermelon mildew resistant strain derived from PI 254744 by providing the resistance marker of watermelon powdery mildew disease according to the present invention.

In addition, by providing the resistance genome region and sequence that can be used to develop various HRM markers in hybrid breeding using PI 254744 in the future, it is possible to easily develop HRM markers in the corresponding nucleotide sequences of other commercial lines to be hybridized It is expected.

Moreover, according to the breeding method of the present invention, it is possible to breed watermelon lines resistant to powdery mildew through crossing using PI 254744 resources as a batch and commercial lines SBB and 45NC as repetitive chines.

Fig. 1 is a schematic diagram summarizing a hybrid pedigree of SBB of commercial watermelon system and PI 254744 resistant to powdery mildew disease.
Figure 2 shows the results of genetic mapping of SB2 and PI254744 hybrid F2 generations.
FIG. 3 is a QTL analysis of the phenotype obtained by the F3 generation resistance test obtained with self-moisture of SBB and PI 254744 hybridization F2.
4 shows the nucleotide sequence of SEQ ID NO: 1 and the position of the SNP in the corresponding nucleotide sequence.
5 shows the nucleotide sequence of SEQ ID NO: 2 and the SNP position in the corresponding nucleotide sequence.
6 shows the nucleotide sequence of SEQ ID NO: 3 and the SNP position in the corresponding nucleotide sequence.
7 is a schematic diagram summarizing the growth of two NILs of the BC4F6 generation obtained by repeating SB 25B and 45 NC repeatedly with PI 254744 as a batch.
FIG. 8 shows the result of genotyping using HRM markers. Each graph represents the genotype, the red graph represents individuals with resistance to foliar resistance (R), the blue graph represents individuals with susceptibility (S), the yellow graph represents hetero (H = R + S) The phenotype represents resistance.

As a result of efforts to develop an HRM marker for the identification of resistance to watermelon (Citrullus lanatus, watermelon), the present inventors determined the resistance genome region by QTL analysis in the commercial SBB line and the F2: third generation of the wild type PI 254744 The genome sequences of the parents of the mating parents were compared with each other to obtain SNPs. The SNPs were confirmed by designing primers that confirmed the phenotype and genotype of all individuals. And the present invention has been completed on the basis thereof.

Hereinafter, the present invention will be described in detail.

The present invention provides a polynucleotide sequence comprising 8-100 contiguous bases comprising a single nucleotide polymorphism (SNP) located in the 553rd base within the nucleotide sequence of SEQ ID NO: 1 or a complementary polynucleotide sequence thereof ; A polynucleotide sequence consisting of 8-100 contiguous bases comprising a single nucleotide polymorphism (SNP) located in position 553 within the nucleotide sequence of SEQ ID NO: 2, or a complementary polynucleotide sequence thereof; And a polynucleotide sequence consisting of 8-100 contiguous bases comprising a single nucleotide polymorphism (SNP) located in the 549th base within the nucleotide sequence of SEQ ID NO: 3, or a complementary polynucleotide sequence thereof Wherein the polynucleotide comprises at least one polynucleotide sequence selected from the group consisting of the polynucleotide sequence of SEQ ID NO: 1 or a complementary polynucleotide sequence thereof.

The nucleotide sequence of SEQ ID NO: 1 to SEQ ID NO: 3 according to the present invention may be a nucleotide sequence existing on the chromosome 2 of the watermelon, preferably 25,800,000 to 27,000,000 bp.

In one embodiment of the present invention, the DNA of the F2 generation individuals obtained by hybridization of the watermelon resistant disease wild type PI 254744 with the susceptible cultivar SBB was extracted, and the gene map was created by GBS analysis. F3 generation (F2 generation (QTL) analysis was carried out through phenotypic tests of the genetically modified plants. The genomic region of PI 254744, which is presumed to be a resistant region in QTL analysis, was determined.

In addition, after the inoculation of the powdery mildew with the fungi in the F3 generation, which is a self-pollution generation of F2, 20 resistant individuals and 20 susceptible individuals were selected, and DNA was extracted from each of these individuals (NGS) were carried out. The SNPs of the isolates obtained from the resistant and susceptible isolates were compared with each other to determine regions showing the greatest SNP difference and frequency, and were estimated as the resistance genome region. The common region of the PI 254744 genome region determined by the above two methods was identified as 25,800,000 - 27,000,000 bp region of chromosome 2 based on the reference chromosome base sequence, and SNPs existing in the corresponding region were obtained.

The SNP marker according to the present invention is characterized in that the base located at the 553rd base in the nucleotide sequence of SEQ ID NO: 1 is T -> C, the 553th base is T -> C in the nucleotide sequence of SEQ ID NO: 2, , It can be judged that the 549th base is G - > A in the nucleotide sequence of the present invention, it is resistant to watermelon powdery mildew. Therefore, by using a primer set capable of amplifying the SNP marker as a molecular marker, DNA can be extracted from a young plant or seed without direct inoculation, and the resistance can be confirmed immediately.

Accordingly, in another aspect of the present invention, there is provided a primer set for discriminating resistance-to-watermelon individuals capable of amplifying the polynucleotide sequence or a complementary polynucleotide sequence thereof, , A primer set consisting of SEQ ID NOS: 4 and 5; A primer set consisting of SEQ ID NOS: 6 and 7; And a set of primers consisting of SEQ ID NOS: 8 and 9.

The term "primer " in the present invention is a nucleic acid sequence having a short free 3 'hydroxyl group, capable of forming base pairs with a complementary template and serving as a starting point for template strand copying ≪ / RTI > The primer can initiate DNA synthesis in the presence of a reagent for polymerization and a different four nucleoside triphosphate at a suitable buffer solution and temperature.

In another embodiment of the present invention, a genotype: phenotype test was performed on each individual (total of 137 individuals) of the F2: 3 isolate population using three primer sets, and as a result, The same HRM results were obtained.

As described above, the primer set according to the present invention can be used for confirming the mutation in the nucleic acid sequence through HRM (High Resolution Melting) analysis. In another embodiment of the present invention, the present invention provides a primer set And provides a PCR kit for HRM analysis for discriminating resistance to watermelon mildew.

In the present invention, "HRM" means that the amplification product DNA bound to the fluorescent reagent is changed in temperature from about 60 ° C to 90 ° C according to the SNP on the DNA present in the PCR region, Means a method of measurement.

Further, as another embodiment of the present invention,

1) performing PCR by mixing the PCR kit with whole DNA samples extracted from watermelon; And

2) performing high resolution melting (HRM) analysis using the amplified sample of step 1).

Meanwhile, in another embodiment of the present invention, a resistance to the powdery mildew disease was obtained after hybridization with the watermelon PI 254744 to obtain a powdery mildew resistant strain. In particular, the molecular markers according to the present invention showed resistance to genetic foliar disease Respectively.

Accordingly, as another embodiment of the present invention, there is provided a method of breeding watermelon having a fungus resistance including the following steps:

(a) obtaining interspecific F1 by interspecific hybridization of resistant rice wild type PI 254744 and cultivated SBB or 45NC;

(b) obtaining a population of BC4F1, which is a species of watermelon powdery mildew resistant species, through crossing the interspecies F1 with SBB or 45NC again (F1 × SBB or F1 × 45NC); And

(c) self-moisturizing the BC4F1.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the following examples.

[Example]

Example 1. Experimental Preparation and Experimental Method

1-1. Preparation of watermelon wild type PI 254744

Of the US Plant Introduction (PI), known as a wild-type resistant to watermelon powdery mildew, PI 254744 was purchased from the US Department of Agriculture USDA-ARS, which was screened through pre-powdery mildew resistance screening for watermelon wild resources, The genotype was fostered as a fixed lineage.

1-2. Determination of resistance to powdery mildew of the watermelon wild-type PI 254744

Since the fungus is a fungus that is parasitic to plants, it is collected from watermelons in a greenhouse in Anseong Chung - Ang University, Gyeonggi - do, and isolated from watermelons and melons and used as an inoculum. At the time of inoculation, the concentration of powdery mildew was 1 × 10 ⁶ spores / mL. The infected leaves were soaked with water and scraped with a brush to adjust the inoculum concentration. The inoculation was sprayed once in the cotyledon spreader and twice every week in the first leafhopper, and the relative humidity was maintained at 90-100% for 3 to 4 days after the inoculation for vinyl infection. The response of the plants to the powdery mildew after inoculation was established by the discrimination index as shown in Table 1, and classified into resistance, moderate resistance and susceptibility.

Discrimination score
(Rating) Resistance / susceptibility determination Disease index 0 Resistance No symptoms at all One Resistance Slightly fainter yellowing 2 Resistance Initiation of yellowing lesion 3 Resistance Sulphated lesions show cotyledon or leaf more than 20% 4 (Moderate) resistance Brown necrosis spot in addition to yellowing lesion 5 (Moderate) resistance 2-3 white spores occur in a circle 6 (Moderate) resistance Less than 20% of white powdery spores 7 sensibility 20-50% white powder spores 8 sensibility White powder spores 50-70% 9 sensibility Whole white spores or plant mortality

Example  2. To identify the watermelon tolerant genomic region QTL  analysis

2-1. Analysis of resistance to powdery mildew in the watermelon wild-type PI 254744

PI 254744 was mated with commercial strain SBB susceptible to powdery mildew, securing a later generation (Fig. 1). As shown in Table 2 below, the F1 plants that were the first generation after mating showed resistance to powdery mildew, and they were separated into F: 2: 3: 1 (resistant: susceptible). Also, in the backcross generation obtained by crossing F1 with the sample SBB and PI 254744, respectively, the results corresponding to Mendel's law of the genetics were obtained. In addition, the resistance of the cotyledons and the main leaf was identical

group Population Observation phenotype Expectant phenotype Degree of freedom
Chi squared
P
R S R S P1 (SBB) 20 20 P2 (PI254744) 20 20 F1 20 15 5 F2 137 100 37 102.75 34.25 One 0.058 0.782 BC1P1 54 26 28 27 27 One 3.34 0.260 BC2P2 52 50 2 52 0 One 0.509 0.495

From the results in Table 2, it can be concluded that the resistance gene possessed by the watermelon wild-type PI 254744 is a single factor complete dominance, which is different from the incomplete dominance result of Arka Manic , It is confirmed that the gene is not the same gene and is a non-allelic gene that is not related to each other.

2-2. Genetic mapping and F2: resistance of 3 groups

(F2: 3 groups) were obtained by self-pollination of F1 obtained by crossing of a specimen (SBB of watermelon system and PI 254744 of a watermelon species) and self-pollination of F2 individuals. DNA was isolated from mature leaves of each F2 generation, and GBS (genotyping by sequencing) was performed for SNP-based gene mapping.

More specifically, the DNA extracted from each F2 individual was cut into ApeKI for GBS, and a DNA sequence (a generic adapter) synthesized with a DNA sequence for identifying each individual and a sequence for sequencing was ligated . In order to perform next generation sequencing (NGS), a sequencing library was constructed using a PCR primer complementary to an adapter sequence. The constructed library was sequenced with Watermelon genomic fragments ligation in Illumina GAII sequencer and single nucleotide polymorphism (SNP) from each F2 was analyzed by bioinformatics software. Based on the obtained SNP information, a linkage map was created using JoinMap (v. 4.1) software, which is shown in Fig.

In addition, 20 F3-derived F3 individuals from each F2: 3 isolate were planted in the pots and inoculated with a powdery mildew. The degree of resistance was scored based on the discrimination index (Table 1) described in Example 1-2, The results are shown in Table 3 below. ≪ tb > < TABLE >

number F3 object number Phenotype test Score Phenotype One F3-12 5.5 R 2 F3-18 6.0 R 3 F3-19 6.0 R 4 F3-31 3.5 R 5 F3-32 3.0 R 6 F3-33 3.0 R 7 F3-36 2.5 R 8 F3-37 5.5 R 9 F3-42 2.0 R 10 F3-43 2.0 R 11 F3-46 3.0 R 12 F3-48 2.0 R 13 F3-58 2.0 R 14 F3-61 1.0 R 15 F3-94 2.0 R 16 F3-99 3.5 R 17 F3-100 2.5 R 18 F3-102 2.5 R 19 F3-105 3.0 R 20 F3-108 2.5 R 21 F3-4 8.0 S 22 F3-23 7.5 S 23 F3-27 7.5 S 24 F3-38 8.5 S 25 F3-41 8.0 S 26 F3-44 9.0 S 27 F3-45 8.5 S 28 F3-47 7.5 S 29 F3-49 8.0 S 30 F3-51 8.0 S 31 F3-54 8.5 S 32 F3-56 9.0 S 33 F3-72 8.5 S 34 F3-76 8.0 S 35 F3-79 8.0 S 36 F3-80 8.5 S 37 F3-84 8.0 S 38 F3-88 8.0 S 39 F3-91 8.5 S 40 F3-118 8.0 S

2-3. Watermelon resistance QTL  analysis

The genetic map information prepared in Example 2-2 and the phenotypes of the disease response (resistance or susceptibility) corresponding to each F2 determined in the F2: 3 group were collected, and the results were collected using the open software Windows QTL Cartographer (ver. 2.5 ) Were used for quantitative trait loci (QTL) analysis.

As a result, as shown in FIG. 3, among the 11 watermelon chromosomes, 25,800,000-27,000,000 bp of the chromosome 2 appeared, and the QTL was tested at the same locus of the resistance locus of the cotyledon and the first locus of resistance . In the QTL region, LOD 19 ~ 21.5 was observed in only one chromosome 2 of the whole chromosome, and it could be limited to a single region of the entire genome.

Example  3. For the identification of resistance to watermelon powdery mildew HRM Marker  Development

3-1. Securing SNP for resistance to watermelon powdery mildew

In order to more specifically compare the SNPs in the resistance genome region identified in Example 2-3, DNAs of PI 254744 and SBB strains were extracted and subjected to NGS analysis using HiSeq 2500 from Illumina ( SNP was obtained by comparison with the sequence of the reference genome (www.icugi.org). In addition, utilizing the SNP results of PI 254744 and SBB strains, different SNPs were searched using Tablet software within the tolerance zone of fungi resistance analyzed by Example 2-3.

As a result, the 553th base in the nucleotide sequence of SEQ ID NO: 1, the 553th nucleotide in the nucleotide sequence of SEQ ID NO: 2, and the SNP located in the 549th nucleotide in the nucleotide sequence of SEQ ID NO: 3 6).

3-2. primer  Set production

A primer set for each SNP was prepared using Primer 3 (http://primer3.ut.ee/), a web-based software centered on the SNP obtained in Example 3-1, and the primer set for each primer set Specific information is shown in Table 4 below.

Primer Sequence mer Set 1
254Pmr_04-F primer AGGATCTTGT GTTACTTCCG GA (SEQ ID NO: 4) 22 254Pmr_04-R primer TTATGATCCA ATTGCTTCCG C (SEQ ID NO: 5) 21 Set 2
254Pmr_05-F primer GGTCGGCCTT GTAATTTCGT (SEQ ID NO: 6) 20 254Pmr_05-R primer AGAACAAACG CTGCACAGTT (SEQ ID NO: 7) 20 Set 3
254Pmr_06-F primer AATGACTCAC CTCCCACAAA TG (SEQ ID NO: 8) 22 254Pmr_06-R primer TGGTCATCCC TAAAGCAATA GTG (SEQ ID NO: 9) 23

Example  4. HRM Marker  Applicability Verification

A genotype: phenotyping test was performed on each individual (total 137 individuals) of the F2: 3 isolate population over 2014 and 2015 using the primer set prepared in Example 3.

As a result, as shown in Table 5 below, it was confirmed that the three sets of primers used showed only one abnormal result in F2-337, and all the individuals showed the same HRM results without any differences among the primers. The difference in F2-337 can be recognized as an experimental error.

On the other hand, phenotypes tested in each of the F2: 3 isolates showed the same phenotype in 2014 and 2015 (some were not tested in 2015 due to insufficient populations), indicating that the environmental impact was very small .

Also, in all 137 tested individuals, the genotype confirmed by phenotype and HRM marker matched. Only in F2-317 one phenotype was susceptible but genotype was resistant. This suggests that the F3-137 progression to test for phenotypes did not lead to self-sufficiency of F2-137.

F2 object number Phenotype test Genotype test year 2014 2015 254Pmr04 254Pmr05 254Pmr06 F2-2 S - ^z) S S S F2-3 S - S S S F2-4 R - H H H F2-6 R - H H H F2-8 R - H H H F2-10 S - S S S F2-12 S - S S S F2-17 R - R R R F2-23 R - R R R F2-25 R - H H H F2-38 R - H H H F2-39 R - H H H F2-56 R - H H H F2-58 R - H H H F2-61 R - H H H F2-62 S - S S S F2-64 R - R R R F2-65 R - H H H F2-67 S - S S S F2-68 R - R R R F2-70 R - H H H F2-71 R - H H H F2-75 S - S S S F2-76 R - R H H F2-79 S - S S S F2-81 R - H H H F2-82 R - H H H F2-83 R - H H H F2-84 R - R R R F2-85 S - S S S F2-86 R - H H H F2-89 R - H H H F2-91 R - R R R F2-92 R - H H H F2-93 R - H H H F2-94 R - H H H F2-97 R - H H H F2-99 R - H H H F2-100 R - R R R F2-104 R - R R R F2-105 R - H H H F2-106 S - S S S F2-107 S - S S S F2-111 R - H H H F2-112 S - S S S F2-113 R - R R R F2-115 S - S S S F2-118 S - S S S F2-121 S - S S S F2-124 S - S S S F2-125 R - R R R F2-301 S S S S S F2-302 S S S S S F2-304 S S S S S F2-305 R R R R R F2-307 S S S S S F2-308 R R R R R F2-309 R R H H H F2-312 R R H H H F2-316 S S S S S F2-317 S S R R R F2-319 R R H H H F2-320 R R R R R F2-321 R R H H H F2-323 S S S S S F2-331 R R H H H F2-332 R R R R R F2-333 R R R R R F2-336 R R H H H F2-337 R R H S H F2-338 S S S S S F2-341 S S S S S F2-342 R R H H H F2-343 R R R R R F2-344 S S S S S F2-345 S S S S S F2-346 R R H H H F2-347 R R H H H F2-348 R R H H H F2-349 R R H H H F2-350 R R R H R F2-351 S S S S S F2-353 R R S S S F2-354 S S S S S F2-356 R R H H H F2-357 R R H H H F2-359 S S S S S F2-360 R R H H H F2-361 R R H H H F2-362 S S S S S F2-363 R R H H H F2-366 R R R R R F2-370 S S S S S F2-372 R R H H H F2-376 R R H H H F2-377 R R R R R F2-378 R R H H H F2-382 R R H H H F2-383 R R R R R F2-388 R R H H H F2-390 S S S S S F2-391 R R H H H F2-393 R R H H H F2-394 R R R R R F2-395 R R H H H F2-396 S S S S S F2-399 R R R R R F2-400 R R H H H F2-402 R R R H H F2-403 R - H H H F2-404 R R R R R F2-405 R R H H H F2-406 R R H H H F2-407 R R H H H F2-408 R R H H H F2-409 R R R R R F2-411 R - R R R F2-412 R R H H H F2-413 S S S S S F2-414 S S S S S F2-417 R R H H H F2-418 S S S S S F2-419 R R H H H F2-421 R S H H H F2-425 R R H H H F2-427 R R H H H F2-430 R R H H H F2-434 R R R R R F2-438 R R R R R F2-439 R R H H H F2-440 R R R R R F2-441 R R R R R F2-442 R R R R R F2-444 R R H H H F2-445 R R H H H F2-447 R R H H H F2-449 R R R H H

(R), susceptibility (S), genotype: resistance (R, H), susceptibility (S)

Example  5. In powdery mildew resistant strains HRM Marker  Applicability Verification

5-1. Myxoid system (NIL; near- isogenic  line)

In order to test the utility of the developed HRM markers, PI 254744 resources were used as a batch, and fungi-resistant fungi resistant NILs were grown using commercially available SBB and 45NC repeats, A schematic procedure of this method is shown in Fig. On the other hand, each NIL was fostered in BC4F6 generation.

5-2. Of the NIL HRM Marker  black

For each of the NILs developed, a resistance to phenotypic resistance was tested.

As a result, as shown in Table 6 and FIG. 8, the NIL individuals were consistent with the genotype results of developed HRM markers and the phenotypic test results of resistance / susceptibility. In addition, we showed 3: 1 (resistance: susceptible) separation ratios corresponding to the Mendel's law in each of the two NIL species and in the subsequent F2 isolates crossed with commercial strain TS susceptible to powdery mildew. , It was shown that resistance genotyping of PI 254744 against future powdery mildew resistant individuals was possible.

F2 (TS x SBB-NIL_BC4F6) F2 (TS x 45NC-NIL_BC4F6) number Phenotype genotype number Phenotype genotype One S S S One R R R 2 R R H 2 R R H 3 S S S 3 S S S 4 S S S 4 S S S 5 R R R 5 S S S 6 R R H 6 R R R 7 S S S 7 R R R 8 S S S 8 R R H 9 R R R 9 S S S 10 R R H 10 R R H 11 S S S 11 S S S 12 R R H 12 R R R 13 S S S 13 S S S 14 R R R 14 S S S 15 R R H 15 S S S 16 S S S 16 R R H 17 R R R 17 S S S 18 R R H 18 R R R

The HRM-utilizing primer sets for genotyping were all 254Pmr04, 254Pmr05, and 254Pmr06, and the genotyping results of the three primer sets were the same.

Thus, the PI 254744-derived powdery mildew resistance-associated molecular markers of the present invention can be successfully used for the development of resistance to watermelon mildew resistance by using PI 254744 as one of the transgenic lines. And that it is possible to distinguish it.

It will be understood by those skilled in the art that the foregoing description of the present invention is for illustrative purposes only and that those of ordinary skill in the art can readily understand that various changes and modifications may be made without departing from the spirit or essential characteristics of the present invention. will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.

<110> Chung-Ang University Industry-Academy Cooperation Foundation <120> Molecular marker for selecting watermelon line or cultivar          resistant to powdery mildew and use thereof <130> MP15-122 <160> 9 <170> KoPatentin 3.0 <210> 1 <211> 1106 <212> DNA <213> Citrullus lanatus <400> 1 tagaagtata tcacttatag actatatagc tgtttttgct ttcactgagt ataggttcag 60 taaaaaaggc aactaaattt ttcttcaaaa aggtgccgtt ataaatatct caaatgtata 120 catcaataat cgtttgtaag ttcaaaacac acttttacta cttttttttt atctctcaat 180 tttgatctca aatattttaa attggtgaat tttgaagaat aagtttaaaa agatctcaac 240 taataaattt attataattta aatataaaat taattaaata tatgacgttt taatatgatt 300 ttagaaaaaa aaaattatat tttttaatat ttcccacttc tctctcttct ttctctactt 360 tttcttcctc ttctctcttg ttctctctcc acatctccat ctccaacatt gttgttacag 420 cacccatctc ttgcgcagcc attccaacac atagatctga aacccagctc cattccaacc 480 aattatgtct actggatcta ctattaggat cttgtgttac ttccggagat actattcaaa 540 attgtatttt ttttctctta ttactatttg atagaaatga aggaacataa agaatacgca 600 gcggaagcaa ttggatcata aacactctaa tttgaaagta aaaacatgct atttttaagc 660 tataaaagag agggtttgaa atacaacatt tgtagcacca aattcttgca aattagcctt 720 tcagttaatc acgaacaaga tatagaccac cacaagtgtc ttccttgcta ttctctggtt 780 ttagaacttg attgtgggat caaattagtg atgaattgaa gggaattttg tgaattgaga 840 gagattgaga gacttttagt tttagtgctt aatttcaaca aattcaagca tcatcattct 900 tcttcatgaa tctcattcat aatttctttt taaagagatt aacatgcaaa aatgagttat 960 catttcaaat tgacacataa tttaagtagg attagtgtca aattgagaag aacacgccta 1020 gcatgcaaaa atgagtactg atttcaagtt gacaccttac ataagtgtga ttagtatcaa 1080 attgagaaca atgcaccaag cctttc 1106 <210> 2 <211> 1106 <212> DNA <213> Citrullus lanatus <400> 2 agatttacgt ttacaacata aaataataat agttgagatc taattttgat aaattatttt 60 accaattgtt acaaaattga aaaaaaaaaa gtgttttaaa aaaaaacaaa aaacataaga 120 agtggtggcc gggaccggcg ggacgagtag aaggacctta tcttttctct cagacgatga 180 actcactacc gtcgcaagcg attcgacctc tttcactctc ttcttcttct tcttcatctt 240 caagctctct ctaccttcgt ttcatctctt catccttccc aatttcccct tatttcaatc 300 cccaatcccc tgtttttgcc gccatttccc gccgactacg acgttccacc atcagaagct 360 gctcctccat caccgccaag ccatcctcgg agatcagaag aacccgccct aaggatgaac 420 ccgattccaa gcttcgggct ctccgtgagt tgttctcgag acccaccatt ggtatcgacg 480 cctatgtaat cccctcacag gacgctcacc aggtcggcct tgtaatttcg ttttctctgg 540 tctcttctgt gttgtaagag cacaaagtca tttcgatgca actgtgcagc gtttgttctt 600 aaggggtttg gaaaattctg agtaattaag actttgtgct gttgaatttt tttttttatc 660 aacgtgttgt gttgatgatg aactcatttg gatgtgttac tgaattgtga aatgtttgtt 720 cttgagggtt tttttttcct ttgttcttgg aaggagaaat atgagtcatc aggtcttcat 780 ttggtaacca ttgggtgtat tttttttttc ttttagttat tgaaaattgt gcttgtttct 840 tcacaatttc ttaaccatgg ttttcataaa ttctgaaaac aaaaaacaag tctacgatgg 900 attctttttt gtgaaatgtt gctcaattgt gcaatgtttg ttcttgaact ttttctgggg 960 gtggggagaa gaaatataga gtaattgcga ttttatgctc ctgaaatttt atggaagatc 1020 tattttcatc gtaatttcgt tatgtctaat ctcttctgtg tcgttagagc ataaagtcat 1080 ttggatgtgt tactcaactg tgcagc 1106 <210> 3 <211> 1098 <212> DNA <213> Citrullus lanatus <400> 3 aaagtaccat acactttaac actataaatt ttacttcatt aatatgtata gactccacaa 60 taatatcagt aggtactgct atactttaaa aaaactcgtt gacatgacaa acaataaatg 120 aaaattttgt ttaaaaaaga aacgaacaag caaattaata ccgcaaaaca ttctctacct 180 aactttttta ttgaattctt aaaattaaat ttaaaaaaaa ttaaaaaaaa aagaaggaaa 240 aagtatactt ttttttattg aattaaacaa aagaaagaaa taaattagaa aatccacttt 300 tctggttcga tgccacctta actgttttcc gcctcacgtt tatttaattt ttttttaatt 360 aaaatattat tttaggcttt aatatatatt cttaattgtc taattttcat cttcgtaatt 420 tcaacaaata aacaaattta gtctatgtac ttttaataaa tttttgagat ttttgtacaa 480 taaccaattc ataagtacat ttatgattaa tgactcacct cccacaaatg tatgaaatct 540 aactttctgg ttacattcga gcgcactatt gctttaggga tgaccatatg taccatagtc 600 ctaacatgat ggtcatccct atcgcgttat tcatttttag agactagttt tttttataga 660 gaaaaccaac cataaactaa tcatagtgac taattttaag attcattcaa agaataagaa 720 ctaaaacttg acattttaaa gtacaaagac tatagaacaa agctaataat ttagtgacca 780 aaatggtatt taaacttaaa tttaaaaaaaattatttta aaatataaga aaatgtatca 840 aaatatttac aaatatagca aaatttcact atctatgtgc aatggaccgc gatagactat 900 tatctatatt tatatgtgtc ataatagaca cggatagtag tctatcgcaa tttattattg 960 tgtatcacag atagattatg atattttacg attatttgta aatattttca acaatcttgt 1020 cattttccta aaacaaaata caataaaact gaataaagtt tttatcaaat ttccttgatg 1080 aaaatttgtc actggata 1098 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> 254Pmr_04-F primer <400> 4 aggatcttgt gttacttccg ga 22 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> 254Pmr_04-R primer <400> 5 ttatgatcca attgcttccg c 21 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 254Pmr_05-F primer <400> 6 ggtcggcctt gtaatttcgt 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 254Pmr_05-R primer <400> 7 agaacaaacg ctgcacagtt 20 <210> 8 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> 254Pmr_06-F primer <400> 8 aatgactcac ctcccacaaa tg 22 <210> 9 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> 254Pmr_06-R primer <400> 9 tggtcatccc taaagcaata gtg 23

Claims (6)

A polynucleotide sequence consisting of 8-100 contiguous bases comprising a single nucleotide polymorphism (SNP) located in position 553 within the nucleotide sequence of SEQ ID NO: 1, or a complementary polynucleotide sequence thereof;
A polynucleotide sequence consisting of 8-100 contiguous bases comprising a single nucleotide polymorphism (SNP) located in position 553 within the nucleotide sequence of SEQ ID NO: 2, or a complementary polynucleotide sequence thereof; And
A polynucleotide sequence consisting of 8-100 contiguous bases comprising a single nucleotide polymorphism (SNP) located in the 549th nucleotide in the nucleotide sequence of SEQ ID NO: 3 or a complementary polynucleotide sequence thereof Wherein the polynucleotide comprises at least one polynucleotide sequence selected from SEQ ID NO: 1 or SEQ ID NO: 2, or a complementary polynucleotide sequence thereof.
A primer set for identifying a pollenucleotide sequence of claim 1 or a resistance to watermelon mildew that can amplify a complementary polynucleotide sequence thereof.
3. The method according to claim 2, wherein the primer set comprises a primer set consisting of SEQ ID NOS: 4 and 5; A primer set consisting of SEQ ID NOS: 6 and 7; And at least one primer set selected from the group consisting of primers set forth in SEQ ID NOs: 8 and 9, in order to identify a watermelon mildew resistant subject.
A PCR kit for HRM analysis for identifying a Watermelon mildew resistant organism comprising the primer set of claim 2 or 3.
(1) performing PCR by mixing the PCR kit of claim 4 with whole DNA samples extracted from watermelon; And
(2) performing a high resolution melting (HRM) analysis using the amplified sample of step (1).
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